Fig 1: KLK10 protects endothelial permeability against thrombin and oscillatory shear (OS).Human aortic endothelial cells (HAECs) were grown to confluency on biotinylated gelatin and were treated with (a, b) rKLK10 (10 ng/ml) or vehicle for 16 hr followed by thrombin (5 U/ml for 30 min), or (c, d) exposed to OS (±5 dynes/cm2) with rKLK10 (10 ng/ml) or vehicle for 24 hr. Endothelial permeability was then measured by the binding of FITC-avidin to the biotinylated gelatin. (b, d) Quantification of endothelial permeability measured as FITC-avidin fluorescence intensity. N = 3 each. Scale bar = 50 µm. All data are represented as mean ± standard error of mean (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA) with Bonferroni correction (b) or paired t-test (d).
Fig 2: Ultrasound-mediated overexpression of Klk10 plasmid inhibits atherosclerosis development.(a) Bioluminescent imaging of Apoe-/- partial carotid ligation (PCL) mice on a high-fat diet injected with luciferase control plasmid or Klk10-luciferase plasmid, measured in photons/second. (b) Gross plaque images of excised carotid arteries and (c) quantification of plaque burden normalized to the percentage of the luciferase control. (d) H&E staining of sections from the left carotid artery (LCA) and right carotid artery (RCA) of mice injected with luciferase control plasmid or Klk10-luciferase plasmid. Scale bar low mag = 250 µm, high mag = 50 µm. (e) Quantification of plaque area measured in µm2. All data are represented as mean ± standard error of mean (SEM). Statistical analyses were performed using paired t-test. N = 11. (f) Sections from the RCA and LCA were coimmunostained with anti-KLK10 (orange) and anti-CD31 (red) antibodies. Blue is DAPI. Arrows indicate the ECs. L is the lumen and Adv is the adventitia. Scale bar = 10 µm. (g) Quantification of endothelial KLK10 fluorescent intensity represented as fold-change normalized to luciferase control. (h) Western blot analysis of KLK10 expression in lung tissue from mice injected with control luciferase plasmid or Klk10 plasmid (Figure 6—source data 1). (i) Quantification of KLK10 expression normalized to GAPDH and luciferase control. Plasma lipid analysis of (j) total cholesterol, (k) triglycerides, (l) high-density lipoprotein (HDL) cholesterol, (m) low-density lipoprotein (LDL) cholesterol, or (n) non-HDL cholesterol. All data are represented as mean ± SEM. Statistical analyses were performed using paired t-test. N = 5. ns = not significant. Figure 6—source data 1.Western blots for KLK10 and GAPDH.
Fig 3: KLK10 inhibits NF?B p65 phosphorylation and nuclear translocation.(a, b) Human aortic endothelial cells (HAECs) were treated with rKLK10 (10 ng/ml) or vehicle for 16 hr followed by TNFa (5 ng/ml for 4 hr). Cell lysates were then collected and analyzed for phosphorylated p65 (p-p65) by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Data are expressed as p-p65 fold-change normalized to GAPDH and vehicle control. N = 3 (Figure 3—source data 1). (c, d) HAECs were treated with rKLK10 (10 ng/ml) or vehicle for 16 hr followed by TNFa (5 ng/ml for 4 hr). Cells were then fixed and immunostained for p65 using anti-p65 antibody. Data are expressed as nuclear p65/total p65, normalized to the vehicle control. N = 6. (e, f) HAECs were treated with rKLK10 (10 ng/ml) or vehicle for 16 hr and exposed to shear for 1 hr. Cells were then fixed and immunostained for p65 using anti-p65 antibody. Data are expressed as nuclear p65/total p65, normalized to the static control. N = 4. All data are represented as mean ± standard error of mean (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA) with Bonferroni correction. Figure 3—source data 1.Western blots for p-p65 NFkB and GAPDH.
Fig 4: KLK10 expression is decreased in human coronary arteries with advanced atherosclerotic plaques.(a) Human coronary artery sections with varying degrees of atherosclerotic lesions were stained with anti-KLK10 antibody (red) and DAPI (blue). Scale bar low mag = 500 µm, scale bar; high mag = 50 µm. Arrows indicate endothelial cells. (b) Consecutive arterial sections from the same patients were stained with anti-CD31 antibody (red) and DAPI (blue). (c) Quantification of endothelial KLK10 fluorescence intensity in lower stage plaques (AHA grades 1–3) and advanced stage plaques (AHA grades 4–6). Data are from 40 different patients. Statistical analyses were performed using unpaired t-test. Mean ± standard error of mean (SEM) (Table 1).
Fig 5: KLK10 expression is suppressed by disturbed flow (d-flow) and elevated by stable flow (s-flow) in endothelial cells (ECs) in vitro and in vivo.(a) Depiction of the partial carotid ligation (PCL) surgery and flow-sensitive regions in the aortic arch: right carotid artery (RCA; s-flow), left carotid artery (LCA; d-flow), greater curvature (GC: s-flow), and lesser curvature (LC; d-flow). Two days following the PCL of C57BL/6J mice, the RCA and LCA were collected for frozen section imaging (b, c) and (d) endothelial-enriched RNA preparation. (b) Confocal images of immunostaining with anti-KLK10 or anti-CD31 antibodies (red) and counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue) are shown. Scale bar = 20 µm. Arrows indicate endothelial cells and L is the lumen. (c) Quantification of endothelial KLK10 fluorescence intensity expressed as fold-change normalized to the RCA. N = 4. (d) Klk10 mRNA was measured in endothelial-enriched RNA from the carotid arteries by quantitative real-time polymerase chain reaction (qPCR). Data are expressed as fold-change normalized to 18s internal control. N = 3–4. (e) Confocal images of en face coimmunostaining of the LC and GC with anti-KLK10 (green) and anti-VE-Cadherin (red) antibody are shown counterstained with DAPI (blue). Scale bar = 10 µm. (f) Quantification of endothelial KLK10 fluorescence intensity expressed as fold-change normalized to the GC. N = 5. (g–j) Human artery endothelial cells (HAECs) subjected to 24 hr of unidirectional laminar shear (LS; 15 dynes/cm2) or oscillatory shear (OS; ± 5 dynes/cm2) were used to measure expression of KLK10 mRNA by qPCR (g), KLK10 protein in cell lysates by western blot (h, i), and KLK10 protein secreted to the conditioned media by ELISA (j). N = 4–6. All data are represented as mean ± standard error of mean (SEM). Statistical analyses were performed using paired t-test (Figure 1—source data 1). (k) Single-cell RNAseq analysis of Klk10 gene transcripts and (l) single-cell ATACseq analysis of Klk10 chromatin accessibility in eight endothelial cell clusters (E1–E8), smooth muscle cells (SMCs), fibroblasts (Fibro), 4 monocytes/macrophages clusters (Mo1–4), dendritic cells (DCs), and T cells (T) in the mouse carotid arteries following 2 days or 2 weeks of the PCL surgery as we recently reported (Andueza et al., 2020). The published datasets (Andueza et al., 2020) were reanalyzed here for the Klk10 gene. E1–E4 clusters represent ECs exposed to s-flow conditions in the RCA. E5 and E7 clusters represent ECs exposed to acute (2 days) d-flow in the LCA. E6 and E8 clusters represent ECs exposed to chronic (2 weeks) d-flow in the LCA. TSS indicates transcription start site. Figure 1—source data 1.Western blots for KLK10 and GAPDH.
Supplier Page from MyBioSource.com for Human Kallikrein 10 ELISA Kit